Amino acid analysis.

Amino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion-exchange chromatography and precolumn derivatization followed by reversed-phase HPLC (RP-HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages.

Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase binding.

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC-electrospray ionization (ESI)-MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3'-kinase (PI3-kinase).

Identification of proteins electroblotted to polyvinylidene difluoride membrane by combined amino acid analysis and bioinformatics: An ABRF multicenter study.

The ABRF amino acid analysis study evaluated the general utility of amino acid analysis (AAA) for identification of proteins after denaturing gel electrophoresis and electroblotting to polyvinylidene difluoride (PVDF) membrane.Thirty-eight participating laboratories analyzed a known control (ovalbumin, 5 microg applied to the gel) and either lysozyme or bovine serum albumin as unknown samples (1-, 5-, and 10-microg amounts applied to the gel). Analyses of the unknowns yielded average compositional errors of approximately 30%, 19%, and 18%, respectively, from the low, intermediate, and higher sample amounts; the ovalbumin control exhibited an approximately 17% average error.

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus.

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known.